1. Field of the Invention
The present invention relates to a method of analyzing DNA adducts, in particular to the method of analyzing ethylated thymidine DNA adducts.
2. Description of the Related Art
Humans are often exposed to various chemicals that will change the structure of DNA to produce excessive DNA adducts, and if these DNA adducts cannot be repaired effectively, mutation or carcinogenesis may occur. The amount of DNA adducts in tissues may reflect a balance between the capability of forming and the capability of repairing the adducts in the tissues when the tissues is chemically damaged. Since DNA adducts participate in carcinogenesis, DNA adducts have been used as a biomarker of carcinogens and cancer risk assessments [1].
Alkylating agents are usually found in our living environment. For example, the smoke of cigarettes contains polycyclic aromatic hydrocarbons (PAHs) and tobacco related nitrosamines become potential alkylating agents after a metabolism takes place by cytochrome P450 enzymes [2, 3]. A direct reaction of non-bulky alkylating agents usually occurs in the smoke of cigarettes. Methylated and ethylated DNA adducts were found in human tissues and urine [4-13]. Unlike the methylated DNA adducts, some researches discovered that the amount of ethylated DNA adducts in smokers is higher than that in nonsmokers [5, 6, 9-13]. The ethylation of DNA may occur on phosphate backbone [14, 15] or on bases of DNA. For example, reports [8-13, 16] show that ethylations are found at positions such as N3 of adenine, O6 and N7 of guanine, and O2, O4, and N3 of thymidine (as shown in FIG. 1). In an ethylated DNA adduct, O6-ethylguanine (O6-edT) and O4-ethylthymidine (O4-edT) will cause miscoding lesions, and researches of animal models discovered that these adducts are related to carcinogenesis [17, 18]. In addition, O6-ethylguanine can be repaired effectively, but O2-ethylthymidine (O2-edT) or O4-ethylthymidine (O4-edT) cannot be repaired effectively [18-22]. Both O2-edT and O4-edT are accumulated in living organisms to cause persistent DNA lesions [18-20]. 3-ethyladenine and N7-ethylguanine can be spontaneous depurinated and can be detected in a smoker's urine [9-11].
In smokers' urine, the average amount of 3-ethyladenine is higher than that of non-smokers [9, 10]. Similarly, the average amount of N7-ethylguanine in smokers' urine and liver is higher than that of non-smokers [11, 12]. The amount of O4-edT in the DNA of cancer patients' lung is directly proportional to the amount of PAH-DNA adducts, showing that these two DNA adducts are formed mainly by the smoke of cigarettes as an exposure source [5]. Further, some researches pointed out that the average amount of N-ethylvaline in smokers' hemoglobin rises significantly than that of non-smokers [23]. From the aforementioned results, we can derive that these ethylated DNA adducts may be produced by the ethylating agents in the smoke of cigarettes.
In an evaluation of the role of the ethylated DNA adducts played in the carcinogenesis, a highly sensitive, specific, qualitative and quantitative method is required to analyze the ethylated DNA adducts in vivo. Gas chromatography with electron impact ionization mass spectrometry (GC-EI/MS) was used by previous studies to analyze 3-ethyladenine in urine [8-10]. However, liquid chromatography together with electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) was used for the analysis of N7-ethylguanine in human urine and liver samples [11, 12].
In a complicated matrix, an appropriate internal standard is generally required for quantifying a trace of analyte accurately. However, an isotopomer with the same structure of the analyte has the same expected physical and chemical properties of the analyte except in the mass spectrometer, so that the isotopomer can be used as an ideal internal standard. The stable isotope-labeled internal standard also can be used as a carrier for carrying a trace of analyte processed with a sample processing procedure. The isotope-labeled standard also can be used to label a signal position of the analyte in a chromatogram and used to find a peak of the analyte from the standard in the complicated chromatogram via the isotope according to the substantially the same retention time.
Up to now, there is no method primarily using the mass spectrometer to analyze ethylated thymidine DNA adducts. Present researches have succeeded detecting and quantifying O4-edT, which induces mutation, in healthy persons' livers [4], cancer patents' lung tissues [5], cells of lower respiratory tracts [6], and smokers' lung tissues [13]. However, the white blood cells of healthy ones have not been used for the detection of O4-edT. In these researches, the detection of O4-edT adopts the 32P-postlabeling technique. Unlike the stable isotope dilution chromatography, internal standards [5, 6, 13] used in the 32P-postlabeling technique have different structures and retention time for the high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) are different from those of the analytes.
In summation, the conventional methods for detecting DNA adducts require a large number of samples, fail to find the distributed positions of the analytes from the chromatogram easily, and fail to detect several adducts at the same time, thus the conventional methods have the drawbacks of wasting labor, materials and examination time, and also have the shortcoming of a low sensitivity. Therefore, the inventor of the present invention designs a method of analyzing ethylated thymidine DNA adducts to overcome the drawbacks of the prior art and enhanced industrial applications.